ASC-deficient non-human mammal

Abstract

To provide a mouse model in which the untranslated region of the Pycard gene is preserved while only the coding region for ASC is deleted.SOLUTION: A genetically modified non-human mammal comprises, in its genome, the DNA that includes (i) a base sequence of the untranslated region of the Pycard gene and does not include (ii) a base sequence encoding the ASC protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a non-human mammal which has the untranslated region of the Pycard gene but is deficient in only the region encoding ASC. [Background technology]

[0002] ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is one of the components of inflammasomes, which are intracellular complexes involved in controlling inflammatory responses to infection and injury. ASC is encoded by the translation region of the Pycard gene. Mice in which ASC has been knocked out by knocking out the entire Pycard gene (including the untranslated region) are known (Non-Patent Document 1: Yamamoto M et al., Genes Cells. 2004; 9(11): 1055-67; ​​Non-Patent Document 2: Mariathasan S, et al., Nature. 2004; 430(6996): 213-8.; Reference 3: Ozoren N, et al., J Immunol.2006; 176(7): 4337-42). However, no mice that lack only the region encoding ASC while retaining the untranslated region of the Pycard gene are known. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Yamamoto M et al., Genes Cells. 2004; 9(11): 1055-67 [Non-Patent Document 2] Mariathasan S, et al., Nature. 2004; 430(6996): 213-8. [Non-Patent Document 3] Ozoren N, et al., J Immunol.2006; 176(7): 4337-42 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention has been made in consideration of these circumstances, and the problem to be solved by the present invention is to provide a mouse that has the untranslated region of the Pycard gene, but is deficient only in the region encoding ASC. [Means for solving the problem]

[0005] As a result of intensive research to solve the above-mentioned problems, the inventors have succeeded in producing a genetically modified non-human mammal whose genome contains DNA that includes the base sequence of the untranslated region of the Pycard gene but does not contain the base sequence encoding the ASC protein, thereby completing the present invention. That is, the present invention is as follows.

[0006] [1] The following DNA: (i) containing the nucleotide sequence of the untranslated region of the Pycard gene; and (ii) DNA that does not contain a nucleotide sequence encoding the ASC protein. A genetically modified non-human mammal comprising in its genome. [2] The animal described in [1] above, wherein the base sequence of the untranslated region of the Pycard gene comprises the base sequences of the 5' untranslated region and the 3' untranslated region of the Pycard gene, the base sequence of the 5' untranslated region comprising the base sequence of SEQ ID NO: 1, and the base sequence of the 3' untranslated region comprising the base sequence of SEQ ID NO: 2. [3] The animal described in [1] above, wherein the DNA contains the base sequence of the 5' untranslated region of the Pycard gene, a start codon, a stop codon, and the base sequence of the 3' untranslated region, in that order, and does not contain a base sequence encoding an ASC protein between the start codon and the stop codon. [4] The animal described in [1] above, wherein the DNA comprises the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, and has at least 90% sequence identity with the base sequence of SEQ ID NO: 3. [5] The animal described in [1] above, wherein the DNA comprises the base sequence of SEQ ID NO: 3. [6] The animal according to [1] above, in which submucosal edema of the colon due to induction of an immune response is suppressed, as compared to a normal non-human mammal. [7] The animal according to [1] above, in which pulmonary inflammatory responses induced by induction of an immune response are not suppressed, as compared with normal non-human mammals. [8] A method for producing an ASC-deficient genetically modified non-human mammal, comprising the steps of deleting a base sequence encoding an ASC protein while maintaining the base sequence of the untranslated region of the Pycard gene in the genome of the non-human mammal, The genetically modified non-human mammal comprises the following DNA: (i) containing the nucleotide sequence of the untranslated region of the Pycard gene; and (ii) DNA that does not contain a nucleotide sequence encoding the ASC protein. In the genome, The method. [9] The method described in [8] above, wherein the step of deleting the base sequence encoding the ASC protein is carried out by inserting DNA containing a stop codon immediately after the start codon of the base sequence encoding the ASC protein in the genome of the non-human mammal. Effect of the Invention

[0007] By using the present invention, it is possible to analyze the function of only the ASC protein, without the need to take into account the function of the untranslated region of the Pycard gene. [Brief description of the drawings]

[0008] [Figure 1-1] [Figure 1A] Schematic diagram of the asc / pycard gene locus, donor DNAs for gRNA1 and gRNA2, and mutated alleles in AD mice. The positions of primers used for PCR are also shown. [Figure 1-2] [Figure 1B] to [Figure 1D] B: A diagram showing the results of confirming the genome-edited sequence by DNA sequencing using the conventional Sanger method. C: A diagram showing the results of mutation-specific PCR using a specific primer set. D: A diagram showing the results of Western blotting of ASC expression in bone marrow and spleen lysates. This was performed on wild-type homozygous mice (asc + / +), AD homozygous mice (asc - / -), and heterozygous mice (asc + / -). Tubulin was used as a control. [Diagram 2] Figures show the results of investigating cytokine production and survival in WT and AD mice. After stimulation with LPS, AD mice survive longer than WT mice, and IL-1β secretion is suppressed. [A and B]: Results of 8-hour incubation of bone marrow cells (2 × 105) with MDP or LPS at the indicated concentrations. IL-1β (A) and TNF-α (B) concentrations in the culture supernatants were measured by ELISA. Results are shown as the mean SD of triplicate cultures. Data are the results of three independent experiments. [C-E]: Results of intraperitoneal injection of 50 μg / g LPS in PBS (black circles) or PBS alone (white circles) in WT and AD mice. Four hours after stimulation with LPS, serum IL-1β (C), TNF-α (D), and IL-6 (E) concentrations were measured by ELISA. Results are plotted and mean values ​​are shown. *p value < 0.05 was considered significant. [F]: Kaplan-Meier survival curve data for WT (asc + / +) mice (n = 8) and AD (asc − / −) mice (n = 7) injected with 50 μg / g LPS were analyzed using the log-rank test (p = 0.0006). [Diagram 3]Figure 1 shows histopathological images of WT mouse tissues after LPS stimulation. Each image shows a representative example from five mice. HE staining images of stomach, small intestine, colon, lung, heart, liver, kidney, spleen, and brain tissues from WT mice at 0, 4, and 12 hours after LPS stimulation are shown. Asterisks indicate submucosal edema. Small arrows indicate capillary dilation. Large arrows indicate congestion. Arrowheads indicate neutrophils. Bars (black lines) in the figure indicate 100 μm (low power view), 25 μm (medium power view), and 5 μm (high power view), respectively. [Figure 4] Figure 1 shows histopathological images of AD mouse tissues after LPS stimulation. Each image shows a representative example from five mice. HE staining images of stomach, small intestine, colon, lung, heart, liver, kidney, spleen, and brain tissues from AD mice at 0, 4, and 12 hours after LPS stimulation are shown. Capillary dilation is indicated by small arrows. Congestion is indicated by large arrows. Neutrophils are indicated by arrowheads. Bars (black lines) in the figure indicate 100 μm (low power view), 25 μm (medium power view), and 5 μm (high power view), respectively. [Diagram 5] 1 shows the results of histopathological comparison of colonic submucosal edema in WT and AD mice after LPS stimulation, with each image showing a representative example from each group. A: HE staining of colon before LPS stimulation (0 hours (0h)) and 12 hours (12h) after LPS stimulation. Bar (black line) = 100 μm. B: Description of the method used to measure the thickness of the colonic mucosa (a), submucosa (b), and muscularis propria (c). The average thickness was the average of a1 + a2 + a3. The average thickness of the submucosa was the average of b1 + b2 + b3. The average thickness of the muscularis propria was the average of c1 + c2 + c3. Bar (black line) = 100 μm. C: The average thickness of the submucosa is shown. D: The average thickness of the submucosa (b) relative to the total thickness of the mucosa / submucosa / muscularis propria (a + b + c) is shown. E: The average thickness of the mucosa is shown. F: The average thickness of the muscularis propria is shown. *p value < 0.05. **p value < 0.01. [Figure 6-1][Figure 6A] and [Figure 6B] Histopathological comparison of acute lung injury in WT and AD mice after LPS stimulation. Each image shows a representative example from each group. A: HE staining of lungs before LPS stimulation (0 h) and 12 h after LPS stimulation. Neutrophils attached to the endothelium are indicated by arrowheads. Bar (black line) = 100 μm. B: Method used to measure the number of neutrophils attached to the endothelium of interstitial capillaries and the average capillary circumference. The total number of neutrophils in the capillaries marked with yellow lines was counted. A total of 34 capillaries were counted and the circumference of each was measured. The number of neutrophils per unit of capillary circumference was calculated. Bar (black line) = 100 μm. [Figure 6-2] [Figure 6C]-[Figure 6E] Histopathological comparison results of acute lung injury in WT and AD mice after LPS stimulation. C: Mean number of neutrophils adhering to the endothelium of interstitial capillaries. D: Mean capillary circumference. E: Number of neutrophils adhering to the endothelium of interstitial capillaries versus circumference of dilated capillaries. Results are expressed as mean ± standard deviation. *p value < 0.05. **p value < 0.01. [Figure 7] Immunohistochemical comparison of IL-1β, TNF-α, and IL-6 expression in the colon of WT and AD mice after LPS stimulation. Formalin-fixed and paraffin-embedded specimens from the colon were stained with hematoxylin and eosin (HE). Serial sections were immunostained with antibodies specific for IL-1β, TNF-α, and IL-6. Negative controls (no primary antibody) are also included. Positive staining for IL-1β, TNF-α, and IL-6 is indicated by single, double, and triple arrows, respectively. Asterisks indicate submucosal edema. Neutrophils are indicated by arrowheads. "0h" indicates before LPS stimulation and "12h" indicates 12 hours after LPS stimulation. AD: ASC-deficient, WT: wild type. Bar (black line) = 100 μm. [Figure 8]Immunohistochemical comparison of IL-1β, TNF-α, and IL-6 expression in the lungs of WT and AD mice after LPS stimulation. Formalin-fixed and paraffin-embedded specimens from lungs were stained with hematoxylin and eosin (HE). Serial sections were immunostained with antibodies specific for IL-1β, TNF-α, and IL-6. A negative control (no primary antibody) is also included. Positive staining for IL-1β, TNF-α, and IL-6 is indicated by arrowheads, black arrows, and red arrows, respectively. "0h" indicates before LPS stimulation, and "12h" indicates 12 hours after LPS stimulation. AD: ASC-deficient, WT: wild type. Bar (black line) = 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below. The following embodiments are merely illustrative for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0010] 1. overview Currently, ASC knockout mice are known, but in all of these mice, the entire Pycard gene (including the untranslated region) is knocked out, and this knockout results in the loss of function of the untranslated region RNA in the Pycard gene. Therefore, when these knockout mice are used to analyze the function of ASC, the knockout function may be mistaken for that of ASC, even though it is actually a function of the untranslated region RNA of the Pycard gene, making it difficult to accurately analyze the function of ASC alone. Furthermore, if pharmaceutical development is carried out under the mistaken belief that the function of the non-translated region RNA of the Pycard gene is a function of ASC, then pharmaceuticals targeting ASC may not have an effect on the function of the non-translated region RNA of the Pycard gene and therefore may not achieve the desired effect. In order to avoid this situation and accurately analyze the function of ASC alone, the present inventors created a genetically modified non-human mammalian animal that contains the untranslated region of the Pycard gene but does not contain a nucleotide sequence encoding the ASC protein. By performing analysis using the non-human mammal of the present invention, it becomes possible to perform accurate functional analysis of only ASC, without taking into consideration the influence of the untranslated region of the Pycard gene. Therefore, the non-human mammal of the present invention is extremely useful for research on ASC and for the development of pharmaceuticals targeting ASC.

[0011] 2. Genetically modified non-human mammals The genetically modified non-human mammal of the present invention comprises in its genome DNA which (i) contains a nucleotide sequence from the untranslated region of the Pycard gene, and (ii) does not contain a nucleotide sequence encoding the ASC protein.

[0012] In the present invention, non-human mammals include, but are not limited to, rodents such as mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, monkeys, etc. In the present invention, non-human mammals are preferably rodents, more preferably mice and rats, but are not limited to these.

[0013] The Pycard gene is a gene that contains a translational region that encodes ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), one of the components of the inflammasome, and contains untranslated regions on the 5' and 3' ends of the translational region. The nucleotide sequences of the Pycard genes of non-human mammals are publicly known, and those skilled in the art can access public gene databases, such as NCBI's GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), to obtain information on the nucleotide sequences of the Pycard genes of various non-human mammals. When using, for example, the Pycard gene of a mouse (eg, C57BL / 6J) as the Pycard gene, the gene information can be obtained by entering the accession number NC_000073.7 on the GenBank web page.

[0014] In the present invention, the "nucleotide sequence of the untranslated region of the Pycard gene" includes the nucleotide sequence of the 5' untranslated region and / or the 3' untranslated region of the Pycard gene. In the present invention, the base sequence of the 5' untranslated region and the base sequence of the 3' untranslated region can be appropriately selected according to the type of non-human mammal selected, and are not limited thereto. For example, when a C57BL / 6J mouse is used as the non-human mammal, the base sequence of the 5' untranslated region can include, but is not limited to, one that contains or consists of the base sequence of SEQ ID NO: 1. In addition, the base sequence of the 3' untranslated region can include, but is not limited to, one that contains or consists of the base sequence of SEQ ID NO: 2. Furthermore, in the present specification, when a C57BL / 6J mouse is used as a non-human mammal, for example, the base sequence encoding normal ASC is shown in SEQ ID NO:11. Furthermore, in the present specification, when a non-human mammal, for example, a C57BL / 6J mouse, is used, a base sequence including all of the base sequences of the 5'-terminal untranslated region, the base sequence encoding ASC, and the 3'-terminal untranslated region is represented by sequence number 12.

[0015] In the present invention, "DNA that does not contain a base sequence encoding the ASC protein" means DNA that does not contain a base sequence encoding the normal ASC protein due to a mutation such as a deletion, substitution, addition, or insertion in the base sequence encoding the normal ASC protein. Such DNA can be prepared, for example, by inserting a stop codon immediately (immediately downstream) of the start codon of the ASC protein, as described below in "3. Method for producing genetically modified non-human mammals," but methods for introducing mutations into the ASC protein are not limited to this. Methods for introducing a mutation into a nucleotide sequence encoding a protein of interest are known, and a person skilled in the art can introduce a mutation into a nucleotide sequence encoding a protein of interest using known methods.

[0016] In the present invention, "DNA that (i) contains the base sequence of the untranslated region of the Pycard gene, and (ii) does not contain a base sequence encoding the ASC protein" includes DNA that contains the base sequence of the 5' untranslated region of the Pycard gene, a start codon, a stop codon, and the base sequence of the 3' untranslated region, in this order, and does not contain a base sequence encoding the ASC protein between the start codon and the stop codon. DNA that "does not contain a base sequence encoding the ASC protein between the start codon and the stop codon" includes, for example, DNA in which a stop codon has been inserted immediately after the start codon of the ASC protein (the stop codon is adjacent to the start codon downstream). Furthermore, in the present invention, "DNA that (i) contains the base sequence of the non-translated region of the Pycard gene, and (ii) does not contain the base sequence encoding the ASC protein" includes DNA that contains the base sequence of SEQ ID NO: 1, the start codon, the stop codon, the base sequence of SEQ ID NO: 4 (the translated region of exon 1 (excluding the start codon)), the base sequence of SEQ ID NO: 5 (exon 2), the base sequence of SEQ ID NO: 6 (the translated region of exon 3), and the base sequence of SEQ ID NO: 2. As described below, these DNAs may further contain a base sequence at a site to which a primer for genotyping binds.

[0017] In the present invention, examples of base sequences in which a stop codon (TAA, TAG or TGA) is inserted immediately after the start codon of the ASC protein (the stop codon is adjacent to the start codon downstream) include those that contain "ATGTAA", "ATGTAG" or "ATGTGA" in the base sequence encoding the ASC protein.

[0018] In the present invention, when the "DNA not containing a base sequence encoding the ASC protein" is a DNA containing a base sequence in which a stop codon has been inserted immediately after the start codon of the ASC protein, it may also contain, in addition to the stop codon, any tag or primer binding site. Examples of such primer binding sites include sites to which a primer for genotyping binds. Examples of the base sequence of the site to which a primer for genotyping binds include, but are not limited to, the base sequence of SEQ ID NO: 7. Examples of the base sequence of the primer include, but are not limited to, those having the following base sequences: ASC_WT_F 5'-CGCCTGCCCACCCCAGAGCCATGGG-3' (SEQ ID NO: 8) ASC_KO_F 5'-CCTCAGAAGAACTCGTCAAGAAGGGGC-3' (SEQ ID NO: 9) ASC_WT / KO_R 5'-TGGCTGCAGCTGCCACAGCTCCAGACT-3' (SEQ ID NO: 10)

[0019] In the present invention, "DNA that (i) contains a base sequence of the untranslated region of the Pycard gene, and (ii) does not contain a base sequence encoding the ASC protein" includes DNA that contains the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, and has at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence of SEQ ID NO: 3. The above DNA also includes one containing the base sequence of SEQ ID NO:3.

[0020] As used herein, "deficiency" of ASC means that a protein having normal ASC function is not expressed or expression is suppressed. Therefore, in the present invention, DNA that (i) contains a nucleotide sequence of the untranslated region of the Pycard gene and (ii) does not contain a nucleotide sequence encoding the ASC protein also includes DNA lacking the entire nucleotide sequence (including introns) encoding the ASC protein (SEQ ID NO: 11).

[0021] In the non-human mammal of the present invention, colonic submucosal edema due to induction of immune response is suppressed compared to normal non-human mammals. Meanwhile, in the non-human mammal of the present invention, pulmonary inflammatory response due to induction of immune response is not suppressed compared to normal non-human mammals. Here, "induction of immune response" can be induced, for example, by an immune stimulant. Examples of immune stimulants include, but are not limited to, LPS. The presence or absence of submucosal edema can be confirmed by immunohistochemical staining, for example, as shown in Figure 5. Furthermore, the presence or absence of pulmonary inflammatory reaction can be evaluated by confirming the production of inflammatory cytokines in the lungs by immunohistochemical staining, as shown in Figure 8 and other figures.

[0022] 3. Method for producing genetically modified non-human mammals The genetically modified non-human mammal of the present invention can be produced, for example, as follows, but is not limited thereto. (1) Design of guide RNA and construction of expression plasmid ASC deficient (AD) mice can be generated using the CRISPR / Cas9 method. First, a guide RNA is designed using a known optimization tool, such as Optimized CRISPR Design web tool (Massachusetts Institute of Technology, Zhang Lab, http: / / crispr.mit.edu / ). Using an optimization tool, design a single guide RNA (sgRNA) targeting the relevant sequence within exon 1 of the mouse Pycard gene. Next, a plasmid expressing both Cas9 and the sgRNA is created.

[0023] (2) Design and synthesis of donor DNA Next, donor DNA is designed. The donor DNA is a single-strand oligo-DNA (ssODN) and can contain (i) a stop codon, (ii) a primer genotyping sequence (a base sequence at a site where a primer for genotyping binds), and (iii) a homologous left arm and a homologous right arm that are identical to the upstream and downstream sequences of the fragment to be inserted into the mouse genome sequence. The donor DNA can be designed to introduce a stop codon into the coding region of exon 1 of the pycard gene. The designed donor DNA can be produced synthetically.

[0024] (3) Generation of ASC-deficient mice Fertilized eggs are isolated from the mice of interest, and then the isolated fertilized eggs are injected with plasmids expressing both Cas9 and sgRNA along with donor DNA. The injected eggs are then cultured and transferred into the oviducts of pseudopregnant mice to generate F0 mutant mice. Once the mutant mice have been identified, they will be crossed with mice of the same strain to produce F1 mice lacking ASC.

[0025] In this way, by inserting DNA containing a stop codon immediately after the start codon of the base sequence encoding the ASC protein in the genome of a non-human mammal, the base sequence encoding the ASC protein can be deleted while maintaining the base sequence of the untranslated region of the Pycard gene in the genome of the non-human mammal, and an ASC-deficient genetically recombinant non-human mammal of the present invention can be produced.

[0026] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0027] 1. Generation of ASC-deficient mice (1) Guide RNA design ASC deficient (ASC deficient (AD)) mice were generated using the CRISPR / Cas9 method. Specifically, a stop codon (TAG) was inserted into the ASC coding region to generate mice lacking expression of the ASC protein encoded by the pycard gene on chromosome 7, as described below. First, we designed the following two single guide RNAs (sgRNAs) targeting relevant sequences within exon 1 of the mouse Pycard gene using the Optimized CRISPR Design web tool (Massachusetts Institute of Technology, Zhang Lab, http: / / crispr.mit.edu / ): pycard-gRNA1: 5'-GGC AUC UCG CCG CCC CA-3' (SEQ ID NO: 13) pycard-gRNA2: 5'-GCG G GGC ACG AGA UGC CAU CC-3' (SEQ ID NO: 14) Next, to prepare a plasmid expressing both Cas9 and the above sgRNA, we used the pCAG-Cas9-gRNA expression vector. pCAG-Cas9-gRNA is a plasmid in which Cas9 expression is controlled by the CAG promoter and gRNA expression is controlled by the U6 promoter. The pycard-gRNA1 and pycard-gRNA2 oligonucleotides designed above were separately ligated to the Bbs I site downstream of the U6 promoter of this pCAG-Cas9-gRNA expression vector to create plasmids (pCAG-Cas9-pycard gRNA1 and pCAG-Cas9-pycard-gRNA2) expressing hCas9 and each of the designed sgRNAs.

[0028] (2) Examination of sequence cleavage efficiency To examine the efficiency of double-stranded breaks in the target sequence induced by sgRNA and hCas9, the pCMV-dasherGxxFP plasmid was constructed. To construct pCMV-dasherGxxFP, we first amplified the N-terminal dasherGFP sequence and the C-terminal dasherGFP sequence (DNA2.0 Inc. Menlo Park CA) and placed them under the control of the CMV promoter. Next, we inserted a genomic fragment (approximately 500 bp) containing exon 1 of the pycard gene between the two dasherGFP fragments. The pCMV-dasherGxxFP plasmid thus prepared was mixed with the pCAG-Cas9-pycard gRNA1 or pCAG-Cas9-pycard-gRNA2 prepared in (1) above, and transfected into HEK293T cells. EGFP fluorescence was observed under a fluorescent microscope 48 hours after transfection. If fluorescence is observed, it indicates that the genomic fragment containing exon 1 of the pycard gene in pCMV-dasherGxxFP was cleaved by pycard-gRNA1 or pycard-gRNA2, and GFP was reconstituted by homologous recombination repair of EGFP.

[0029] (3) Design and synthesis of donor DNA Donor DNA was designed. The donor DNA is a single-strand oligo-DNA (ssODN) that contains (i) a stop codon, (ii) a primer genotyping sequence (the base sequence of the site where the primer for genotyping binds), and (iii) a 60 bp homologous left arm and a 60 bp homologous right arm that are identical to the upstream and downstream sequences of the fragment to be inserted into the mouse genome sequence (Figure 1A). The donor DNA was designed to introduce a stop codon into the coding region of exon 1 of the pycard gene. In addition, donor DNAs for pycard-gRNA1 and pycard-gRNA2 were designed (ibid.). The designed donor DNA was synthesized and manufactured by Ultramer oligos Integrated DNA Technologies.Inc.

[0030] (4) Generation of ASC-deficient mice Fertilized eggs were isolated from C57BL / 6N mice, and 1-2 pl (5 ng / μL) of pCAG-Cas9-pycard-gRNA1 plasmid or pCAG-Cas9-pycard-gRNA2 plasmid was injected into the isolated fertilized eggs together with donor DNA ssODN for pycard-gRNA1 or donor DNA ssODN for pycard-gRNA2. The injected eggs were cultured and transferred to the oviducts of pseudopregnant mice to generate F0 mutant mice. To identify mutant mice, a genomic region including exon 1 of the pycard gene was amplified from the genomic DNA of each F0 mouse using the following primer set: A-GGA ... target F: 5'-CTC CAC CTA GTT TCT TCA GCC CC-3' (SEQ ID NO: 15) target R: 5'-GTA GAC GAA CAGGG GAC ACA CTC ACC-3' (SEQ ID NO: 16) Twenty-eight mice were obtained from eggs injected with pCAG-Cas9-pycard-gRNA1, of which seven mice had a stop codon inserted by homology directed repair, and nine mice were obtained from eggs injected with pCAG-Cas9-pycard-gRNA2, of which one mouse had a stop codon inserted by homology directed repair.

[0031] Six F0 mice (F0-8, F0-10, F0-11, F0-16, F0-18, and F0-19) were bred with C57BL / 6N mice to obtain F1, and the insertion of a stop codon in exon 1 of the pycard gene was analyzed in the F1 by direct sequencing as described above.

[0032] As a result, the allele containing the inserted stop codon was transmitted to the germline of F1 mice derived from F0-16, F0-18, and F0-19 mice, and gene-deficient mouse lines were established. The gene-deficient mice were produced by Transgenic Co., Ltd. (Fukuoka, Japan). Mice from F0-16 and F0-18 were used for further studies.

[0033] All mice used in the examples herein were maintained under specific pathogen-free (SPF) conditions, and all experiments were performed in accordance with international and Ehime University guidelines for animal experiments under protocols approved by the Ehime University Animal Care Unit Committee (No. 37U4-16). C57BL / 6 mice used in the examples herein were purchased from CLEA Japan (Tokyo, Japan). C57BL / 6 WT mice (asc + / +) and ASC-deficient mice (AD mice (asc - / -)) were housed in standard cages with paper chips under a 12-hour light / dark cycle and constant ambient temperature (22°C) and humidity (45%). The number of mice housed was four per cage. All animals had free access to tap water and food. For LPS administration, each mouse was given 50 mg / kg LPS with or without PBS, and then monitored by researchers every 4 hours for 7 days.

[0034] 2. reagent The LPS from Escherichia coli (serotype O111:B4, phenol-chloroform-petroleum ether extract, catalog number L2630) and muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) (a component of bacterial cell wall peptidoglycan): catalog number A9519) used in the examples herein were purchased from Sigma (St. Louis, MO). The monoclonal rat anti-mouse ASC antibody used in the experiments was previously obtained (Ozoren N, et al., J Immunol. 2006; 176(7): 4337-42).

[0035] 3. Measurement of cytokine production from bone marrow cells Bone marrow cells used in the examples herein were isolated from mice and cultured in 48-well flat-bottom plates (BD Biosciences, San Jose, CA) containing RPMI 1640 supplemented with 10% FBS. Cells (final cell density: 1 × 10 6 Cells (100 / ml) were treated with or without MDP or LPS for 8 h at 37°C / 5% CO2. IL-1β and tumor necrosis factor (TNF)-α concentrations in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences).

[0036] 4. immunohistochemistry Immunohistochemistry was performed using rabbit polyclonal anti-IL-1β (26405-1-AP) (Abcom, Cambridge, United Kingdom; ab205924). Rabbit polyclonal anti-TNF-α (26405-1-AP) and rabbit polyclonal anti-IL-6 (21865-1-AP) were purchased from Proteintech (Rosemont, IL, USA). Sections were obtained from formalin-fixed, paraffin-embedded tissues. Sections (3-5 μm thick) were deparaffinized in xylene and rehydrated in a dilute solution of ethanol. Endogenous peroxidase activity was blocked by adding 0.3% H2O2 in methanol for 30 min. Prior to immunostaining, antigen retrieval was performed by microwave treatment of tissue sections in 10 mmol / L Tris-HCl buffer (pH 8.0) containing 1 mmol / L ethylenediaminetetraacetic acid (EDTA). Sections were blocked with 1% normal bovine serum in 50 mmol / L Tris-buffered saline (TBS) (pH 7.6) and reacted with primary antibodies diluted in blocking buffer. Binding was detected using EnVision+Rabbit / HRP (Dako, Carpinteria, CA, USA), and positive signals were developed with diaminobenzidine tetrahydrochloride (DAB). Tissue sections were counterstained with hematoxylin and mounted with Entellan new (Sigma-Aldrich, St. Louis, MO, USA).

[0037] 5. statistical analysis Statistical analysis was performed using GraphPad Prism Version 9.4.1 (GraphPad Software, San Diego, CA). All results are shown as the mean ± standard deviation from three independent experiments. EXAMPLES

[0038] result (1) Generation of AD mice First, we generated AD mice by inserting a stop codon (TAG) after the initiation codon (ATG) in the coding region of the pycard gene (Figure 1A). ATG G) was confirmed by Sanger sequencing of DNA from wild-type (WT) (asc + / +) mice (FIG. 1B). We also confirmed that a stop codon (TAG) was successfully inserted after the initiation codon (ATG) in the AD homozygous mice (Fig. 1B; middle panel). The sequences of WT mice and AD homozygous mice overlapped in the heterozygous mice (Fig. 1B; lower panel). The genotypes of each mouse were distinguishable by PCR using specific primer sets (Fig. 1C and Table 1). Western blot analysis revealed that endogenous ASC protein was expressed in the spleen and bone marrow of WT mice (asc + / +) but not in AD mice (asc - / -) (Fig. 1D). ASC protein was also expressed in the heterozygous mice (asc + / -), although to a lesser extent than in WT mice (Fig. 1D). The AD mice were born at the expected Mendelian ratio. From the above, it was demonstrated that AD mice can be produced in this example.

[0039] (2) Investigation into cytokine (IL-1β, TNF-α) secretion by bone marrow cells in AD mice To confirm the deficiency of ASC protein, we compared IL-1β and TNF-α secretion from AD and WT bone marrow cells in the presence of LPS or muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in AD mice (Figures 2A and 2B). The ASC protein is essential for IL-1β processing and activation. We found that bone marrow cells from AD mice did not secrete IL-1β (<20 pg / mL) when incubated with either 0.1 or 1.0 μg / mL LPS for 8 h. In contrast, bone marrow cells from WT mice secreted significant amounts of IL-1β under the same conditions (Fig. 2A). On the other hand, both WT and AD mouse bone marrow cells secreted TNF-α when incubated with 0.1 or 1.0 μg / mL LPS for 8 hours (Figure 2B). In contrast, neither AD nor WT cells secreted IL-1β or TNF-α when incubated with 10 or 5.0 μg / mL MDP for 8 hours (Figures 2A and 2B). MDP is known not to stimulate inflammasomes that contain ASC as a component, and was used as a negative control in this example. These results suggest that IL-1β production is suppressed, whereas TNF-α production is not inhibited, in bone marrow cells from AD mice, indicating that the ASC protein is functionally defective.

[0040] (3) Examination of serum concentrations of cytokines (IL-1β, TNF-α, and IL-6) stimulated with LPS Both WT and AD mice were intraperitoneally administered LPS (50 μg / g body weight) or PBS, and serum concentrations of IL-1β, TNF-α, and IL-6 were measured 4 hours later. The values ​​in AD mice were compared with those in WT mice. As a result, there was no difference in serum IL-1β concentrations between PBS-administered AD mice and PBS-administered WT mice. In contrast, serum IL-1β concentrations in LPS-stimulated AD mice were significantly lower than those in WT mice (Figure 2C). On the other hand, there was no difference in serum TNF-α and IL-6 concentrations between AD mice and WT mice, regardless of whether they were stimulated with LPS (Figures 2D and 2E). These results showed that when stimulated with LPS, AD mice had lower serum IL-1β expression levels compared to WT mice, but serum TNF-α and IL-6 expression levels were no different from those of WT mice.

[0041] (4) Examination of survival time of AD mice in LPS-induced sepsis Upon LPS challenge, 8 of 8 WT mice died within 24 hours. In contrast, 1 of 7 AD mice died within 24 hours, but 3 of 7 mice survived for more than 72 hours. However, 4 of 7 AD mice eventually died (Figure 2F). Kaplan-Meier analysis showed that AD mice survived significantly longer than WT mice after LPS-induced sepsis (p = 0.0006; Log-rank test) (Figure 2F). These results demonstrated that AD mice had a longer survival time after LPS-induced sepsis than WT mice.

[0042] (5) Histological analysis of tissues from LPS-induced septic AD mice To identify tissues most susceptible to injury from LPS-induced sepsis, we performed histological analysis of tissues from WT and AD mice. We examined hematoxylin and eosin (HE)-stained tissues (stomach, small intestine, colon, lung, heart, liver, kidney, spleen, and brain) before and 4 and 12 hours after LPS injection (Figures 3 and 4).

[0043] As a result, congestion (indicated by large arrows) and adhesion of neutrophils (indicated by arrowheads) to the capillary endothelium began to appear in the lungs of both WT and AD mice 4 hours after LPS injection (Figures 3 and 4). At least capillary dilation (indicated by small arrows) appeared in the heart, liver, kidney, and spleen of both WT and AD mice 4 hours after LPS injection (Figures 3 and 4). Congestion (indicated by large arrows) and recruitment of neutrophils (indicated by arrowheads), which are characteristics of acute inflammation, were observed more in the interstitial regions of each tissue 12 hours after LPS injection (Figures 3 and 4). In particular, strong submucosal edema (indicated by asterisks) was observed in the colon of WT mice after LPS injection (Figure 3). This appeared 4 hours after LPS injection, but was not seen in AD mice (Figure 4). The histological findings in WT and AD mice are summarized in Tables 1 and 2 below, respectively.

[0044] [Table 1] [Table 2] As described above, by comparing the histopathological findings in multiple tissues between WT and AD mice, we were able to identify specific ASC-dependent histopathological features. Specifically, results from WT mice showed acute inflammation (catarrhal inflammation) in the gastrointestinal tissue, with dilated capillaries associated with interstitial edema without neutrophil recruitment, whereas these features were largely absent in AD mice (Figures 3 and 4).

[0045] (6) Colonic submucosal edema in AD mice As mentioned above, colonic submucosal edema was more severe in WT mice than in AD mice 12 h after LPS stimulation (Figure 5A). To quantitatively compare submucosal edema in WT and AD mice, we measured the thickness of the submucosa at three points within the severe edema area and averaged these values ​​(Figure 5B). The average thickness of the submucosa in WT mice (95.1 ± 10.0 μm) was significantly greater than that in AD mice (11.4 ± 3.7 μm) 12 h after LPS stimulation (Figure 5C). These results indicate that ASCs are involved in LPS-induced intestinal edema.

[0046] (7) Examination of acute interstitial inflammation in the lungs of WT and AD mice In both WT and AD mice, numerous neutrophils adhered to the endothelium of dilated interstitial capillaries around the alveoli in the lungs 4 h after LPS injection, and these neutrophils were recruited to the interstitial space 12 h after LPS injection (Figures 3 and 4). To quantitatively compare alveolar inflammation in WT and AD mice (Figure 6A), we counted the number of neutrophils adhering to the endothelium in the interstitial region 12 hours after LPS stimulation (Figure 6B). The average number of neutrophils adhering to the endothelium in the interstitial region was 6.58 ± 1.37 cells / capillary in WT mice and 6.95 ± 2.33 cells / capillary in AD mice (Figure 6C). The mean perimeter of dilated capillaries in the interstitial region was 155.5 ± 18.0 μm in WT mice and 159.0 ± 18.5 μm in AD mice (Figure 6D). The mean number of neutrophils versus the perimeter of dilated capillaries was 0.043 ± 0.006 cells / μm and 0.042 ± 0.009 cells / μm, respectively (Figure 6E). The mean number of neutrophils adhering to the endothelium of interstitial capillaries, the mean capillary circumference, and the number of neutrophils adhering to the endothelium of interstitial capillaries relative to the circumference of dilated capillaries were significantly different between LPS-treated WT mice and PBS-treated WT mice, and between LPS-treated AD mice and PBS-treated AD mice, but not between WT and AD mice (Figure 6C-E). The results showed that there were no significant differences between WT and AD mice in the mean number of neutrophils adhering to the endothelium of interstitial capillaries, the mean capillary circumference, and the number of neutrophils adhering to the endothelium of interstitial capillaries relative to the circumference of dilated capillaries.

[0047] (8) Examination of the expression of IL-1β, TNF-α, and IL-6 in the lungs and colons of WT and AD mice To determine whether the expression of IL-1β, TNF-α, and IL-6 was associated with colonic submucosal edema and / or acute lung injury, immunohistochemical analysis was performed. In the colon, positive IL-1β staining was widely observed in the interstitial edema tissue of the colonic submucosa (indicated by arrows) in WT mice 12 h after LPS stimulation, whereas it was positive or slightly nonpositive in the submucosa of AD mice ( Fig. 7 ). Positive TNF-α (indicated by two arrows) and IL-6 (indicated by three arrows) staining was observed in the crypt epithelium in both WT and AD mice before LPS stimulation (0 h), whereas most of the TNF-α (indicated by two arrows) and IL-6 (indicated by three arrows) staining was positive in the crypt and glandular epithelium 12 h after LPS stimulation (Figure 7). In the lungs, positive IL-β staining was observed in several neutrophils (indicated by arrowheads) that had infiltrated into the alveolar interstitium in both WT and AD mice 12 h after LPS stimulation, whereas none were observed before LPS stimulation (0 h) (Figure 8). Positive TNF-α (black arrow) staining was observed in the alveolar epithelium of both WT and AD mice 12 h after LPS stimulation, but not before LPS stimulation (0 h). Positive IL-6 staining was observed in the alveolar epithelium 12 h after LPS stimulation, and less positive staining was observed in the alveolar epithelium before LPS stimulation (Figure 8). These results indicate that the effects of ASC deficiency vary from tissue to tissue. In particular, IL-1β expression was limited to a small population of neutrophils (arrowheads) even 12 hours after LPS stimulation in the lung alveolar interstitial tissue. This suggests that the contribution of inflammasomes in the lung is limited. On the other hand, TLR4 downstream cytokines TNF-α (black arrow) and IL-6 (red arrow) were both more highly expressed in alveolar endothelial cells 12 hours after LPS stimulation than before LPS stimulation (Figure 8). These results indicate that acute lung injury accompanied by neutrophil infiltration is relatively weakly associated with ASCs. [Industrial Applicability]

[0048] By using the present invention, it is possible to analyze the function of the ASC protein alone, without the need to take into account the function of the untranslated region of the Pycard gene. [Sequence List Free Text]

[0049] SEQ ID NOs: 4, 7 to 10, and 13 to 16: synthetic DNA

Claims

1. The following DNA: (i) containing the nucleotide sequence of the untranslated region of the Pycard gene; and (ii) DNA that does not contain a nucleotide sequence encoding the ASC protein. A genetically modified non-human mammal comprising in its genome.

2. The animal described in claim 1, wherein the base sequence of the untranslated region of the Pycard gene comprises the base sequences of the 5' untranslated region and the 3' untranslated region of the Pycard gene, the base sequence of the 5' untranslated region comprising the base sequence of SEQ ID NO: 1, and the base sequence of the 3' untranslated region comprising the base sequence of SEQ ID NO:

2.

3. The animal described in claim 1, wherein the DNA contains the base sequence of the 5' untranslated region of the Pycard gene, a start codon, a stop codon, and a base sequence of the 3' untranslated region, in that order, and does not contain a base sequence encoding an ASC protein between the start codon and the stop codon.

4. The animal described in claim 1, wherein the DNA comprises the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2, and has at least 90% sequence identity with the base sequence of SEQ ID NO:

3.

5. The animal described in claim 1, wherein the DNA comprises the base sequence of SEQ ID NO:

3.

6. The animal of claim 1, in which submucosal edema of the colon due to induction of an immune response is suppressed, as compared to a normal non-human mammal.

7. The animal of claim 1, in which pulmonary inflammatory responses due to induction of an immune response are not suppressed, as compared to normal non-human mammals.

8. A method for producing an ASC-deficient genetically modified non-human mammal, comprising the steps of deleting a base sequence encoding an ASC protein while maintaining the base sequence of the untranslated region of the Pycard gene in the genome of the non-human mammal, The genetically modified non-human mammal comprises the following DNA: (i) containing the nucleotide sequence of the untranslated region of the Pycard gene; and (ii) DNA that does not contain a nucleotide sequence encoding the ASC protein. In the genome, The method.

9. The method according to claim 8, wherein the step of deleting the base sequence encoding the ASC protein is carried out by inserting DNA containing a stop codon immediately after the start codon of the base sequence encoding the ASC protein in the genome of the non-human mammal.

Images